Antisense inhibiton of human G-alpha-12 expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of G-alpha-12. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding G-alpha-12. Methods of using these compounds for modulation of G-alpha-12 expression and for treatment of diseases associated with expression of G-alpha-12 are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of G-alpha-12. In particular, this invention relates toantisense compounds, particularly oligonucleotides, specificallyhybridizable with nucleic acids encoding human G-alpha-12. Sucholigonucleotides have been shown to modulate the expression ofG-alpha-12.

BACKGROUND OF THE INVENTION

A vast majority of biologically active molecules including growthfactors, cytokines, neurotransmitters and hormones transduce signals viaspecific cell-surface receptors. Some of these receptors are thencoupled to heterotrimeric GTP-binding proteins (G proteins) which, uponactivation, relay signals to a variety of cellular effectors includingat least four phospholipase C (PLC) variants and adenylyl cyclases.

G proteins mediate external signals by forming heterotrimers consistingof an alpha, beta and gamma subunit. Several isoforms of each subunithave been identified and therefore, through subunit heterogeneity, Gproteins effectively integrate multiple signaling cascades. The alphasubunits of G proteins contain the GTP binding site and intrinsiccatalytic GTPase activity. Based on sequence similarity and function,these subunits have been classified into four major groups; Gs, whichstimulate adenylyl cyclases; Gi, which inhibit adenylyl cyclases; Gq,which activate PLC isoforms and G12/13, which mediate pathwaysassociated with cell growth and differentiation (Hamm, J. Biol. Chem.,1998, 273, 669-672).

G-alpha-12 is a member of the G12/13 subfamily of G proteins and isactivated by a variety of stimuli. It has been shown to participate inseveral kinase cascades including MAPK, ERK and JNK (Jho et al., J.Biol. Chem., 1997, 272, 24468-24474; Voyno-Yasenetskaya et al., J. Biol.Chem., 1996, 271, 21081-21087) as well as being phosphorylated byprotein kinase C (Kozasa and Gilman, J. Biol. Chem., 1996, 271,12562-12567). Consequently, G-alpha-12 has been shown to participate incellular processes as diverse as mitogenesis (Mitsui et al., J. Biol.Chem., 1997, 272, 4904-4910), regulation of pH (Lin et al., J. Biol.Chem., 1996, 271, 22604-22610) and platelet activation (Collins et al.,Oncogene, 1997, 15, 595-600).

The primary function of G-alpha-12, however, is believed to be themediation of cell differentiation and growth. Therefore themanifestations of altered G-alpha-12 expression, specifically as itrelates to the development of cancer, have been the most broadlyinvestigated.

Cellular transformation and acquisition of the metastatic phenotype arethe two main changes normal cells undergo during the progression tocancer and these processes are believed to be linked to the expressionof G-alpha-12.

G-alpha-12 was first isolated from a soft tissue sarcoma cDNA libraryand overexpression of the protein was shown to be sufficient to inducecellular transformation (Chan et al., Mol. Cell. Biol., 1993, 13,762-768).

In studies of NIH3T3 and Rat-1 fibroblasts, constitutively active mutantforms of G-alpha-12 were shown to induce transformation as indicated byincreased proliferation, loss of contact inhibition, focus formation andincreased DNA synthesis (Voyno-Yasenetskaya et al., Oncogene, 1994, 9,2559-2565).

Results from studies in human embryonic kidney cells demonstrated thatconstitutive activation of G-alpha-12 stimulated RhoA-dependantphosphorylation of p125 focal adhesion kinase, paxillin and p130Crk-associated substrate, all of which have been implicated in theregulation of proliferation and transformation (Needham and Rozengurt,J. Biol. Chem., 1998, 273, 14626-14632).

Finally, studies using both antisense vectors expressing a 43 basefragment of mouse G-alpha-12 in antisense orientation and constitutivelyactive forms of G-alpha-12 to investigate retinoic acid-stimulateddifferentiation of P19 mouse embryonal carcinoma cells found thatG-alpha-12 activity was essential to the differentiation process (Jho etal., J. Biol. Chem., 1997, 272, 24468-24474; Jho and Malbon, J. Biol.Chem., 1997, 272, 24461-24467).

Currently, there are no known therapeutic agents which effectivelyinhibit the synthesis of G-alpha-12 and strategies aimed at inhibitingor investigating G-alpha-12 function have involved the use ofantibodies, mutant forms of the protein which are constitutively activeand antisense expression vectors.

These strategies are untested as therapeutic protocols and consequentlythere remains a long felt need for additional agents capable ofeffectively inhibiting G-alpha-12 function. Therefore, antisenseoligonucleotides may provide a promising new pharmaceutical tool for theeffective and specific modulation of G-alpha-12 expression.

SUMMARY OF THE INVENTION

The present invention is directed to antisense compounds, particularlyoligonucleotides, which are targeted to a nucleic acid encodingG-alpha-12, and which modulate the expression of G-alpha-12.Pharmaceutical and other compositions comprising the antisense compoundsof the invention are also provided. Further provided are methods ofmodulating the expression of G-alpha-12 in cells or tissues comprisingcontacting said cells or tissues with one or more of the antisensecompounds or compositions of the invention. Further provided are methodsof treating an animal, particularly a human, suspected of having orbeing prone to a disease or condition associated with expression ofG-alpha-12 by administering a therapeutically or prophylacticallyeffective amount of one or more of the antisense compounds orcompositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric antisense compounds,particularly oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding G-alpha-12, ultimately modulating theamount of G-alpha-12 produced. This is accomplished by providingantisense compounds which specifically hybridize with one or morenucleic acids encoding G-alpha-12. As used herein, the terms "targetnucleic acid" and "nucleic acid encoding G-alpha-12" encompass DNAencoding G-alpha-12, RNA (including pre-mRNA and mRNA) transcribed fromsuch DNA, and also cDNA derived from such RNA. The specifichybridization of an oligomeric compound with its target nucleic acidinterferes with the normal function of the nucleic acid. This modulationof function of a target nucleic acid by compounds which specificallyhybridize to it is generally referred to as "antisense". The functionsof DNA to be interfered with include replication and transcription. Thefunctions of RNA to be interfered with include all vital functions suchas, for example, translocation of the RNA to the site of proteintranslation, translation of protein from the RNA, splicing of the RNA toyield one or more mRNA species, and catalytic activity which may beengaged in or facilitated by the RNA. The overall effect of suchinterference with target nucleic acid function is modulation of theexpression of G-alpha-12. In the context of the present invention,"modulation" means either an increase (stimulation) or a decrease(inhibition) in the expression of a gene. In the context of the presentinvention, inhibition is the preferred form of modulation of geneexpression and mRNA is a preferred target.

It is preferred to target specific nucleic acids for antisense."Targeting" an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding G-alpha-12. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5'-AUG (in transcribed mRNAmolecules; 5'-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the "AUG codon," the "startcodon" or the "AUG start codon". A minority of genes have a translationinitiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, theterms "translation initiation codon" and "start codon" can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, "start codon" and "translationinitiation codon" refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding G-alpha-12, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or"stop codon") of a gene may have one of three sequences, i.e., 5'-UAA,5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAGand 5'-TGA, respectively). The terms "start codon region" and"translation initiation codon region" refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5' or 3') from a translationinitiation codon. Similarly, the terms "stop codon region" and"translation termination codon region" refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5' or 3') from a translationtermination codon.

The open reading frame (ORF) or "coding region," which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5' untranslatedregion (5'UTR), known in the art to refer to the portion of an mRNA inthe 5' direction from the translation initiation codon, and thusincluding nucleotides between the 5' cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3' untranslated region (3'UTR), known in the art to refer to theportion of an mRNA in the 3' direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3' end of an mRNA or corresponding nucleotides onthe gene. The 5' cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5'-most residue of the mRNA via a 5'-5'triphosphate linkage. The 5' cap region of an mRNA is considered toinclude the 5' cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5' cap region may also be a preferred targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as "introns," which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as "exons" and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, "hybridization" means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. "Complementary," as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, "specifically hybridizable" and "complementary" are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotides have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans. In the context of this invention, the term"oligonucleotide" refers to an oligomer or polymer of ribonucleic acid(RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This termincludes oligonucleotides composed of naturally-occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally-occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for nucleic acidtarget and increased stability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 30 nucleobases.Particularly preferred are antisense oligonucleotides comprising fromabout 8 to about 30 nucleobases (i.e. from about 8 to about 30 linkednucleosides). As is known in the art, a nucleoside is a base-sugarcombination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to either the2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides,the phosphate groups covalently link adjacent nucleosides to one anotherto form a linear polymeric compound. In turn the respective ends of thislinear polymeric structure can be further joined to form a circularstructure, however, open linear structures are generally preferred.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3'to 5' phosphodiester linkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3'-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3'-5' linkages, 2'-5' linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular --CH₂ --NH--O--CH₂ --, --CH₂--N(CH₃)--O--CH₂ -- [known as a methylene (methylimino) or MMIbackbone], --CH₂ --O--N(CH₃)--CH₂ --, --CH₂ --N(CH₃)--N(CH₃)--CH₂ -- and--O--N(CH₃)--CH₂ --CH₂ -- [wherein the native phosphodiester backbone isrepresented as --O--P--O--CH₂ --] of the above referenced U.S. Pat. No.5,489,677, and the amide backbones of the above referenced U.S. Pat. No.5,602,240. Also preferred are oligonucleotides having morpholinobackbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2' position: OH; F; O--, S--, or N-alkyl; O--, S--, or N-alkenyl;O--, S-- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyland alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ toC₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n) O]_(m)CH₃, O(CH₂)_(n) OCH₃, O(CH₂)_(n) NH₂, O(CH₂)_(n) CH₃, O(CH₂)_(n) ONH₂,and O(CH₂)_(n) ON[(CH₂)_(n) CH₃)]₂, where n and m are from 1 to about10. Other preferred oligonucleotides comprise one of the following atthe 2' position: C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN,CF₃, OCF₃, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2'-methoxyethoxy (2'--O--CH₂ CH₂ OCH₃,also known as 2'--O--(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2'-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ ON(CH₃)₂ group, also known as 2'-DMAOE, as described in exampleshereinbelow.

Other preferred modifications include 2'-methoxy (2'--O--CH₃),2'-aminopropoxy (2'--OCH₂ CH₂ CH₂ NH₂) and 2'-fluoro (2'-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3' position of the sugar on the 3'terminal nucleotide or in 2'-5' linked oligonucleotides and the 5'position of 5' terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as "base") modifications or substitutions. As used herein,"unmodified" or "natural" nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2'-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference, and U.S. Pat. No. 5,750,692, which is commonly owned withthe instant application and also herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. "Chimeric"antisense compounds or "chimeras," in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term "prodrug" indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 to Imbach et al.

The term "pharmaceutically acceptable salts" refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., "Pharmaceutical Salts," J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a "pharmaceutical addition salt"includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of G-alpha-12 is treated by administering antisense compoundsin accordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingG-alpha-12, enabling sandwich and other assays to easily be constructedto exploit this fact. Hybridization of the antisense oligonucleotides ofthe invention with a nucleic acid encoding G-alpha-12 can be detected bymeans known in the art. Such means may include conjugation of an enzymeto the oligonucleotide, radiolabelling of the oligonucleotide or anyother suitable detection means. Kits using such detection means fordetecting the level of G-alpha-12 in a sample may also be prepared.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2'-O-methoxyethyl modification are believed to beparticularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other. In general, emulsions may be eitherwater-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueousphase is finely divided into and dispersed as minute droplets into abulk oily phase the resulting composition is called a water-in-oil (w/o)emulsion. Alternatively, when an oily phase is finely divided into anddispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil-in-water (o/w) emulsion. Emulsions maycontain additional components in addition to the dispersed phases andthe active drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories--surfactants, fatty acids,bile salts, chelating agents, and non-chelating non-surfactants (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92).Each of these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term "liposome" means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include "sterically stabilized" liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂ 15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the "head") provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p.92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or"surface-active agents") are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term "bile salts"includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, "carrier compound" or"carrier" can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4' isothiocyano-stilbene-2,2'-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a "pharmaceutical carrier" or"excipient" is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include, but are not limitedto, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin,bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 1206-1228). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. See, generally, The Merck Manual of Diagnosis and Therapy,15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and46-49, respectively). Other non-antisense chemotherapeutic agents arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Numerous examples of antisensecompounds are known in the art. Two or more combined compounds may beused together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀ s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and2'-alkoxy Amidites

2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham Mass. or GlenResearch, Inc. Sterling Va.). Other 2'-O-alkoxy substituted nucleosideamidites are prepared as described in U.S. Pat. No. 5,506,351, hereinincorporated by reference. For oligonucleotides synthesized using2'-alkoxy amidites, the standard cycle for unmodified oligonucleotideswas utilized, except the wait step after pulse delivery of tetrazole andbase was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2'-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2'-Fluoro Amidites

2'-Fluorodeoxyadenosine amidites

2'-fluoro oligonucleotides were synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2'-deoxy-2'-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2'-alpha-fluoro atom is introduced by a S_(N) 2-displacement of a2'-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3',5'-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5'-dimethoxytrityl-(DMT) and5'-DMT-3'-phosphoramidite intermediates.

2'-Fluorodeoxyguanosine

The synthesis of 2'-deoxy-2'-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5'-DMT- and5'-DMT-3'-phosphoramidites.

2'-Fluorouridine

Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by themodification of a literature procedure in which2,2'-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5'-DMT and 5'-DMT-3'phosphoramidites.

2'-Fluorodeoxycytidine

2'-deoxy-2'-fluorocytidine was synthesized via amination of2'-deoxy-2'-fluorouridine, followed by selective protection to giveN4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were used toobtain the 5'-DMT and 5'-DMT-3'phosphoramidites.

2'-O-(2-Methoxyethyl) modified amidites

2'-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2'-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.).

2'-O-Methoxyethyl-5-methyluridine

2,2'-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃ CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂ Cl₂ /acetone/MeOH (20:5:3)containing 0.5% Et₃ NH. The residue was dissolved in CH₂ Cl₂ (250 mL)and adsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃ CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂ SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃ NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by TLC by first quenching the TLC sample with the addition ofMeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃ CN (700 mL) and set aside. Triethylamine (189 mL,1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃ CN (1L), cooled to -5° C. and stirred for 0.5 h using an overhead stirrer.POCl₃ was added dropwise, over a 30 minute period, to the stirredsolution maintained at 0-10° C., and the resulting mixture stirred foran additional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

A solution of3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄ OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, TLC showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/hexane (1:1) containing 0.5% Et₃ NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amidite

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂ Cl₂ (1 L) Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂ Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2'-O-(Aminooxyethyl) Nucleoside Amidites and2'-O-(Dimethylaminooxyethyl) Nucleoside Amidites

2'-(Dimethylaminooxyethoxy) nucleoside amidites

2'-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the artas 2'-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5'-O-tert-Butyldiphenylsilyl-O² -2'-anhydro-5-methyluridine

O² -2'-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L)and brine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to -10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 mL) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.

5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5'-O-tert-Butyldiphenylsilyl-O² -2'-anhydro-5-methyluridine (149 g,0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added withmanual stirring. The reactor was sealed and heated in an oil bath untilan internal temperature of 160° C. was reached and then maintained for16 h (pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine

5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried over P₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5'-O-tert-butyldiphenylsilyl-2'-O-[(2-Formadoximinooxy)ethyl]-5-methyluridine

2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂ Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at -10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂ Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂ SO₄. The solution was concentrated to get2'-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂ Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂ SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂ SO₄ and evaporated to dryness . The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂ Cl₂ to get5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2'-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂ Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂ Cl₂ to get 2'-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine

2'-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P₂ O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4'-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂ Cl₂ (containing a fewdrops of pyridine) to get5'-O-DMT-2'-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂ O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹ -tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂ SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2'-(Aminooxyethoxy) nucleoside amidites

2'-(Aminooxyethoxy) nucleoside amidites [also known in the art as2'-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2'-O-aminooxyethyl guanosine analog may be obtained by selective2'-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2'-O-(2-ethylacetyl) diaminopurine riboside along with a minoramount of the 3'-O-isomer. 2'-O-(2-ethylacetyl) diaminopurine ribosidemay be resolved and converted to 2'-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection proceduresshould afford 2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosineand2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

Example 2

Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P=O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P=S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethyl-hydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P=O or P=S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4

PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the "gap" segment oflinked nucleosides is positioned between 5' and 3' "wing" segments oflinked nucleosides and a second "open end" type wherein the "gap"segment is located at either the 3' or the 5' terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas "gapmers" or gapped oligonucleotides. oligonucleotides of the secondtype are also known in the art as "hemimers" or "wingmers".

[2'-O-Me]--[2'-deoxy]--[2'-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and2'-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA portion and5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for 5' and 3' wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2'-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2' positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to 1/2 volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[2'-O-(2-Methoxyethyl)]--[2'-deoxy]--[2'-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2'-O-(2-methoxyethyl)]--[2'-deoxy]--[-2'-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2'-O-methyl chimeric oligonucleotide, with thesubstitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methylamidites.

[2'-O-(2-Methoxyethyl)Phosphodiester]--[2'-deoxyPhosphorothioate]--[2'-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2'-O-(2-methoxyethyl phosphodiester]--[2'-deoxyphosphorothioate]--[2'-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2'-O-methyl chimeric oligonucleotide with the substitution of2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures an d sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹ P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

Oligonucleotide Synthesis--96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄ OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

Oligonucleotide Analysis--96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

Cell culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing four cell types are provided for illustrative purposes, butother cell types can be routinely used.

T-24 Cells:

The transitional cell bladder carcinoma cell line T-24 was obtained fromthe American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cellswere routinely cultured in complete McCoy's 5A basal media (Gibco/LifeTechnologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum(Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units permL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence. Cells were seeded into96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/wellfor use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

Treatment with Antisense Compounds:

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™ 1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired oligonucleotide at a final concentration of150 nM. After 4 hours of treatment, the medium was replaced with freshmedium. Cells were harvested 16 hours after oligonucleotide treatment.

Example 10

Analysis of Oligonucleotide Inhibition of G-alpha-12 Expression

Antisense modulation of G-alpha-12 expression can be assayed in avariety of ways known in the art. For example, G-alpha-12 mRNA levelscan be quantitated by, e.g., Northern blot analysis, competitivepolymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-timequantitative PCR is presently preferred. RNA analysis can be performedon total cellular RNA or poly(A)+mRNA. Methods of RNA isolation aretaught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley& Sons, Inc., 1993. Northern blot analysis is routine in the art and istaught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc.,1996. Real-time quantitative (PCR) can be conveniently accomplishedusing the commercially available ABI PRISM™ 7700 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions. Other methods of PCR arealso known in the art.

G-alpha-12 protein levels can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA or fluorescence-activated cell sorting (FACS).Antibodies directed to G-alpha-12 can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12

Total RNA Isolation

Total mRNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC™ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

Example 13

Real-time Quantitative PCR Analysis of G-alpha-12 mRNA Levels

Quantitation of G-alpha-12 mRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE or FAM, obtained from either Operon Technologies Inc.,Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5' end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3' end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3' quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5'-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular (six-second) intervals bylaser optics built into the ABI PRISM™ 7700 Sequence Detection System.In each assay, a series of parallel reactions containing serialdilutions of mRNA from untreated control samples generates a standardcurve that is used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension). G-alpha-12 probes and primers weredesigned to hybridize to the human G-alpha-12 sequence, using publishedsequence information (GenBank accession number L01694, incorporatedherein as SEQ ID NO:1).

For G-alpha-12 the PCR primers were: forward primer:CTCAAGGGCTCAAGGGTTCTT (SEQ ID NO: 2) reverse primer:CAGGAACATCCCATGCTTCTC (SEQ ID NO: 3) and the PCR probe was:FAM-AAGCTTGGCATTCCTTGGCAGTATTCTGA-TAMRA (SEQ ID NO: 4) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye.

For GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC (SEQID NO: 5) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 6)and the PCRprobe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3' (SEQ ID NO: 7) whereJOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescentreporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) isthe quencher dye.

Example 14

Northern Blot Analysis of G-alpha-12 mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST "B" Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.).

Membranes were probed using QUICKHYB™ hybridization solution(Stratagene, La Jolla, Calif.) using manufacturer's recommendations forstringent conditions with a G-alpha-12 specific probe prepared by PCRusing the forward primer CTCAAGGGCTCAAGGGTTCTT (SEQ ID NO: 2) and thereverse primer CAGGAACATCCCATGCTTCTC (SEQ ID NO: 3). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA(Clontech, Palo Alto, Calif.). Hybridized membranes were visualized andquantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3(Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDHlevels in untreated controls.

Example 15

Antisense Inhibition of G-alpha-12 Expression-PhosphorothioateOligodeoxynucleotides

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human G-alpha-12 RNA,using published sequences (GenBank accession number L01694, incorporatedherein as SEQ ID NO: 1). The oligonucleotides are shown in Table 1.Target sites are indicated by nucleotide numbers, as given in thesequence source reference (Genbank accession no. L01694), to which theoligonucleotide binds. All compounds in Table 1 areoligodeoxynucleotides with phosphorothioate backbones (internucleosidelinkages) throughout. The compounds were analyzed for effect onG-alpha-12 mRNA levels by quantitative real-time PCR as described inother examples herein. Data are averages from two experiments. Ifpresent, "N.D." indicates "no data".

                  TABLE 1                                                         ______________________________________                                        Inhibition of G-alpha-12 mRNA levels by phosphorothioate                        oligodeoxynucleotides                                                                          TARGET            % In- SEQ ID                               ISIS# REGION SITE SEQUENCE hibition NO.                                     ______________________________________                                        20656 Coding   9        ctgagggtccgcaccacc                                                                       0     8                                      20657 Coding 17 ggcagcggctgagggtcc 32 9                                       20658 Coding 21 agcaggcagcggctgagg 7 10                                       20659 Coding 115 gtcgatgtcgcggctacg 0 11                                      20660 Coding 125 ccagcagcgcgtcgatgt 71 12                                     20661 Coding 167 gcaggatcttcaccaggc 7 13                                      20662 Coding 170 ggatgatgcgcatctgct 0 14                                      20663 Coding 186 cagcagcaggatcttcac 8 15                                      20664 Coding 198 gtggacttgccgctctcg 0 16                                      20665 Coding 200 acgtggacttgccgctct 68 17                                     20666 Coding 209 gcttgaggaacgtggact 69 18                                     20667 Coding 217 gcgcatctgcttgaggaa 18 19                                     20669 Coding 235 ctcgcggccgtggatgat 1 20                                      20670 Coding 245 tctggtcgaactcgcggc 30 21                                     20671 Coding 251 gcgccttctggtcgaact 28 22                                     20672 Coding 259 ctccagcagcgccttctg 0 23                                      20673 Coding 269 tgtcgcggaactccagca 21 24                                     20674 Coding 279 tcgaagatggtgtcgcgg 2 25                                      20675 Coding 288 aggatgttgtcgaagatg 23 26                                     20676 Coding 296 agcccttgaggatgttgt 6 27                                      20677 Coding 309 acaagaacccttgagccc 26 28                                     20678 Coding 317 gtgcatcaacaagaaccc 11 29                                     20679 Coding 325 cttatctcgtgcatcaac 49 30                                     20680 Coding 344 actgccaaggaatgccaa 0 31                                      20681 Coding 354 ttttcagaatactgccaa 37 32                                     20682 Coding 366 ccatgcttctcattttca 51 33                                     20683 Coding 377 tcaggaacatcccatgct 21 34                                     20684 Coding 386 cgaaggccatcaggaaca 0 35                                      20685 Coding 400 ccccgccttgttctcgaa 15 36                                     20686 Coding 438 gggacgtacagctggaag 47 37                                     20687 Coding 447 ctcagggccgggacgtac 23 38                                     20688 Coding 452 gtgcgctcagggccggga 28 39                                     20689 Coding 467 cagaatccctccagagtg 63 40                                     20690 Coding 476 ccctgatgccagaatccc 61 41                                     20691 Coding 484 gaaagcctccctgatgcc 62 42                                     20692 Coding 500 actcgcttctccggctga 15 43                                     20693 Coding 530 ggaagtacttcaccgact 0 44                                      20694 Coding 540 aagttgtccaggaagtac 56 45                                     20695 Coding 548 tccggtccaagttgtcca 12 46                                     20696 Coding 552 ccgatccggtccaagttg 62 47                                     20697 Coding 563 aattcagctggccgatcc 41 48                                     20698 Coding 574 actaggaaagtaattcag 53 49                                     20699 Coding 586 gatatcttgcttactagg 63 50                                     20700 Coding 610 cttggtggctttcctagc 0 51                                      20701 Coding 619 cacaattcccttggtggc 0 52                                      20702 Coding 630 aagtcatgctccacaatt 36 53                                     20703 Coding 639 ttaataacgaagtcatgc 27 54                                     20704 Coding 644 tcttcttaataacgaagt 52 55                                     20705 Coding 667 cacatccaccatcttaaa 0 56                                      20706 Coding 679 ccgctggccgcccacatc 53 57                                     20707 Coding 703 ctggaaccacttctggcg 11 58                                     20708 Coding 714 ccgtcgaagcactggaac 20 59                                     20709 Coding 736 catgaacaggatggacgt 50 60                                     20710 Coding 747 ctggaggagaccatgaac 0 61                                      20711 Coding 757 gtcgtactcgctggagga 61 62                                     20712 Coding 764 ggacctggtcgtactcgc 58 63                                     20713 Coding 771 tccatgaggacctggtcg 21 64                                     20714 Coding 784 ggtgcgcctgtcctccat 67 65                                     20715 Coding 793 cagccggttggtgcgcct 75 66                                     20716 Coding 807 ttcatggactccaccagc 43 67                                     20717 Coding 813 aagatgttcatggactcc 67 68                                     20718 Coding 821 tggtctcgaagatgttca 47 69                                     20719 Coding 831 ttgttgacgatggtctcg 27 70                                     20720 Coding 847 gttgaagaagagcttgtt 67 71                                     20721 Coding 858 atgatggagacgttgaag 0 72                                      20722 Coding 866 ggaagagaatgatggaga 0 73                                      20723 Coding 880 gtccatcttgttgaggaa 32 74                                     20724 Coding 889 caccaggaggtccatctt 25 75                                     20725 Coding 910 gctcacggtcttcacctt 0 76                                      20726 Coding 923 agtgcttcttgatgctca 38 77                                     20727 Coding 937 cctgaagtccgggaagtg 15 78                                     20728 Coding 994 cctgtcgaagcactggac 66 79                                     20729 Coding 1006 gttccgtctcttcctgtc 64 80                                    20730 Coding 1028 ggtggaagagtggcttgc 35 81                                    20731 Coding 1038 gtggtgaagtggtggaag 13 82                                    20732 Coding 1066 gaagcggacgttctcggt 0 83                                     20733 Coding 1076 catggaacacgaagcgga 43 84                                    20734 Coding 1106 ggttctcctgcaggatgg 7 85                                     20735 Coding 1122 agcatgatgtccttcagg 71 86                                  ______________________________________                                    

As shown in Table 1, SEQ ID NOs 9, 12, 17, 18, 21, 30, 32, 33, 37, 40,41, 42, 45, 47, 48, 49, 50, 53, 55, 57, 60, 62, 63, 65, 66, 67, 68, 69,71, 74, 77, 79, 80, 81, 84 and 86 demonstrated at least 30% inhibitionof G-alpha-12 expression in this assay and are therefore preferred.

Example 16

Antisense Inhibition of G-alpha-12 Expression-Phosphorothioate 2'-MOEGapmer Oligonucleotides

In accordance with the present invention, a second series ofoligonucleotides targeted to human G-alpha-12 were synthesized. Theoligonucleotide sequences are shown in Table 2. Target sites areindicated by nucleotide numbers, as given in the sequence sourcereference (Genbank accession no. L01694), to which the oligonucleotidebinds.

All compounds in Table 2 are chimeric oligonucleotides ("gapmers") 18nucleotides in length, composed of a central "gap" region consisting often 2'-deoxynucleotides, which is flanked on both sides (5' and 3'directions) by four-nucleotide "wings". The wings are composed of2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P=S) throughout the oligonucleotide.Cytidine residues in the 2'-MOE wings are 5-methylcytidines.

Data were obtained by real-time quantitative PCR as described in otherexamples herein and are averaged from two experiments. If present,"N.D." indicates "no data".

                  TABLE 2                                                         ______________________________________                                        Inhibition of G-alpha-12 mRNA levels by chimeric phosphorothioate              oligodeoxynucleotides having 2'-MOE wings and a deoxy gap                                       TARGET            % In- SEQ ID                                                                         ISIS# REGION SITE SEQUENCE                                                   hibition NO.                       ______________________________________                                        21061 Coding   9        ctgagggtccgcaccacc                                                                       64    8                                      21062 Coding 17 ggcagcggctgagggtcc 53 9                                       21063 Coding 21 agcaggcagcggctgagg 61 10                                      21064 Coding 115 gtcgatgtcgcggctacg 90 11                                     21065 Coding 125 ccagcagcgcgtcgatgt 0 12                                      21066 Coding 167 gcaggatcttcaccaggc 72 13                                     21073 Coding 170 ggatgatgcgcatctgct 71 14                                     21067 Coding 186 cagcagcaggatcttcac 59 15                                     21069 Coding 198 gtggacttgccgctctcg 93 16                                     21070 Coding 200 acgtggacttgccgctct 70 17                                     21071 Coding 209 gcttgaggaacgtggact 49 18                                     21072 Coding 217 gcgcatctgcttgaggaa 72 19                                     21074 Coding 235 ctcgcggccgtggatgat 77 20                                     21075 Coding 245 tctggtcgaactcgcggc 90 21                                     21076 Coding 251 gcgccttctggtcgaact 84 22                                     21077 Coding 259 ctccagcagcgccttctg 75 23                                     21078 Coding 269 tgtcgcggaactccagca 25 24                                     21079 Coding 279 tcgaagatggtgtcgcgg 30 25                                     21080 Coding 288 aggatgttgtcgaagatg 77 26                                     21081 Coding 296 agcccttgaggatgttgt 85 27                                     21082 Coding 309 acaagaacccttgagccc 57 28                                     21083 Coding 317 gtgcatcaacaagaaccc 87 29                                     21084 Coding 325 cttatctcgtgcatcaac 69 30                                     21085 Coding 344 actgccaaggaatgccaa 20 31                                     21086 Coding 354 ttttcagaatactgccaa 0 32                                      21087 Coding 366 ccatgcttctcattttca 34 33                                     21088 Coding 377 tcaggaacatcccatgct 6 34                                      21089 Coding 386 cgaaggccatcaggaaca 27 35                                     21090 Coding 400 ccccgccttgttctcgaa 50 36                                     21091 Coding 438 gggacgtacagctggaag 0 37                                      21092 Coding 447 ctcagggccgggacgtac 91 38                                     21093 Coding 452 gtgcgctcagggccggga 28 39                                     21094 Coding 467 cagaatccctccagagtg 0 40                                      21095 Coding 476 ccctgatgccagaatccc 74 41                                     21096 Coding 484 gaaagcctccctgatgcc 84 42                                     21097 Coding 500 actcgcttctccggctga 31 43                                     21098 Coding 530 ggaagtacttcaccgact 77 44                                     21099 Coding 540 aagttgtccaggaagtac 52 45                                     21100 Coding 548 tccggtccaagttgtcca 64 46                                     21101 Coding 552 ccgatccggtccaagttg 40 47                                     21102 Coding 563 aattcagctggccgatcc 52 48                                     21103 Coding 574 actaggaaagtaattcag 0 49                                      21104 Coding 586 gatatcttgcttactagg 33 50                                     21105 Coding 610 cttggtggctttcctagc 63 51                                     21106 Coding 619 cacaattcccttggtggc 79 52                                     21107 Coding 630 aagtcatgctccacaatt 48 53                                     21108 Coding 639 ttaataacgaagtcatgc 59 54                                     21109 Coding 644 tcttcttaataacgaagt 48 55                                     21110 Coding 667 cacatccaccatcttaaa 49 56                                     21111 Coding 679 ccgctggccgcccacatc 92 57                                     21112 Coding 703 ctggaaccacttctggcg 30 58                                     21113 Coding 714 ccgtcgaagcactggaac 75 59                                     21114 Coding 736 catgaacaggatggacgt 23 60                                     21115 Coding 747 ctggaggagaccatgaac 64 61                                     21116 Coding 757 gtcgtactcgctggagga 0 62                                      21117 Coding 764 ggacctggtcgtactcgc 0 63                                      21118 Coding 771 tccatgaggacctggtcg 60 64                                     21119 Coding 784 ggtgcgcctgtcctccat 86 65                                     21120 Coding 793 cagccggttggtgcgcct 48 66                                     21121 Coding 807 ttcatggactccaccagc 70 67                                     21122 Coding 813 aagatgttcatggactcc 51 68                                     21123 Coding 821 tggtctcgaagatgttca 24 69                                     21124 Coding 831 ttgttgacgatggtctcg 65 70                                     21125 Coding 847 gttgaagaagagcttgtt 54 71                                     21126 Coding 858 atgatggagacgttgaag 48 72                                     21127 Coding 866 ggaagagaatgatggaga 37 73                                     21128 Coding 880 gtccatcttgttgaggaa 54 74                                     21129 Coding 889 caccaggaggtccatctt 4 75                                      21130 Coding 910 gctcacggtcttcacctt 72 76                                     21131 Coding 923 agtgcttcttgatgctca 56 77                                     21132 Coding 937 cctgaagtccgggaagtg 24 78                                     21133 Coding 994 cctgtcgaagcactggac 87 79                                     21134 Coding 1006 gttccgtctcttcctgtc 64 80                                    21135 Coding 1028 ggtggaagagtggcttgc 78 81                                    21136 Coding 1038 gtggtgaagtggtggaag 44 82                                    21137 Coding 1066 gaagcggacgttctcggt 61 83                                    21138 Coding 1076 catggaacacgaagcgga 20 84                                    21139 Coding 1106 ggttctcctgcaggatgg 77 85                                    21140 Coding 1122 agcatgatgtccttcagg 65 86                                  ______________________________________                                    

As shown in Table 2, SEQ ID NOs 8, 9, 10, 11, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 26, 27, 28, 29, 30, 36, 38, 41, 42, 44, 45, 46, 47,48, 51, 52, 53, 54, 55, 56, 57, 59, 61, 64, 65, 66, 67, 68, 70, 71, 72,74, 76, 77, 79, 80, 81, 82, 83, 85 and 86 demonstrated at least 40%inhibition of G-alpha-12 expression in this experiment and are thereforepreferred.

Example 17

Western Blot Analysis of G-alpha-12 Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to G-alpha-12 is used,with a radiolabelled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands are visualizedusing a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 86                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 1146                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1)..(1146)                                                    - - <400> SEQUENCE: 1                                                         - - atg tcc ggg gtg gtg cgg acc ctc agc cgc tg - #c ctg ctg ccg gcc        gag       48                                                                    Met Ser Gly Val Val Arg Thr Leu Ser Arg Cy - #s Leu Leu Pro Ala Glu            1               5 - #                 10 - #                 15              - - gcc ggc ggg gcc cgc gag cgc agg gcg ggc ag - #c ggc gcg cgc gac gcg           96                                                                       Ala Gly Gly Ala Arg Glu Arg Arg Ala Gly Se - #r Gly Ala Arg Asp Ala                        20     - #             25     - #             30                  - - gag cgc gag gcc cgg agg cgt agc cgc gac at - #c gac gcg ctg ctg gcc          144                                                                       Glu Arg Glu Ala Arg Arg Arg Ser Arg Asp Il - #e Asp Ala Leu Leu Ala                    35         - #         40         - #         45                      - - cgc gag cgg cgc gcg gtc cgg cgc ctg gtg aa - #g atc ctg ctg ctg ggc          192                                                                       Arg Glu Arg Arg Ala Val Arg Arg Leu Val Ly - #s Ile Leu Leu Leu Gly                50             - #     55             - #     60                          - - gcg ggc gag agc ggc aag tcc acg ttc ctc aa - #g cag atg cgc atc atc          240                                                                       Ala Gly Glu Ser Gly Lys Ser Thr Phe Leu Ly - #s Gln Met Arg Ile Ile            65                 - # 70                 - # 75                 - # 80       - - cac ggc cgc gag ttc gac cag aag gcg ctg ct - #g gag ttc cgc gac acc          288                                                                       His Gly Arg Glu Phe Asp Gln Lys Ala Leu Le - #u Glu Phe Arg Asp Thr                            85 - #                 90 - #                 95              - - atc ttc gac aac atc ctc aag ggc tca agg gt - #t ctt gtt gat gca cga          336                                                                       Ile Phe Asp Asn Ile Leu Lys Gly Ser Arg Va - #l Leu Val Asp Ala Arg                       100      - #           105      - #           110                  - - gat aag ctt ggc att cct tgg cag tat tct ga - #a aat gag aag cat ggg          384                                                                       Asp Lys Leu Gly Ile Pro Trp Gln Tyr Ser Gl - #u Asn Glu Lys His Gly                   115          - #       120          - #       125                      - - atg ttc ctg atg gcc ttc gag aac aag gcg gg - #g ctg cct gtg gag ccg          432                                                                       Met Phe Leu Met Ala Phe Glu Asn Lys Ala Gl - #y Leu Pro Val Glu Pro               130              - #   135              - #   140                          - - gcc acc ttc cag ctg tac gtc ccg gcc ctg ag - #c gca ctc tgg agg gat          480                                                                       Ala Thr Phe Gln Leu Tyr Val Pro Ala Leu Se - #r Ala Leu Trp Arg Asp           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - tct ggc atc agg gag gct ttc agc cgg aga ag - #c gag ttt cag ctg        ggg      528                                                                    Ser Gly Ile Arg Glu Ala Phe Ser Arg Arg Se - #r Glu Phe Gln Leu Gly                          165  - #               170  - #               175              - - gag tcg gtg aag tac ttc ctg gac aac ttg ga - #c cgg atc ggc cag ctg          576                                                                       Glu Ser Val Lys Tyr Phe Leu Asp Asn Leu As - #p Arg Ile Gly Gln Leu                       180      - #           185      - #           190                  - - aat tac ttt cct agt aag caa gat atc ctg ct - #g gct agg aaa gcc acc          624                                                                       Asn Tyr Phe Pro Ser Lys Gln Asp Ile Leu Le - #u Ala Arg Lys Ala Thr                   195          - #       200          - #       205                      - - aag gga att gtg gag cat gac ttc gtt att aa - #g aag atc ccc ttt aag          672                                                                       Lys Gly Ile Val Glu His Asp Phe Val Ile Ly - #s Lys Ile Pro Phe Lys               210              - #   215              - #   220                          - - atg gtg gat gtg ggc ggc cag cgg tcc cag cg - #c cag aag tgg ttc cag          720                                                                       Met Val Asp Val Gly Gly Gln Arg Ser Gln Ar - #g Gln Lys Trp Phe Gln           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - tgc ttc gac ggg atc acg tcc atc ctg ttc at - #g gtc tcc tcc agc        gag      768                                                                    Cys Phe Asp Gly Ile Thr Ser Ile Leu Phe Me - #t Val Ser Ser Ser Glu                          245  - #               250  - #               255              - - tac gac cag gtc ctc atg gag gac agg cgc ac - #c aac cgg ctg gtg gag          816                                                                       Tyr Asp Gln Val Leu Met Glu Asp Arg Arg Th - #r Asn Arg Leu Val Glu                       260      - #           265      - #           270                  - - tcc atg aac atc ttc gag acc atc gtc aac aa - #c aag ctc ttc ttc aac          864                                                                       Ser Met Asn Ile Phe Glu Thr Ile Val Asn As - #n Lys Leu Phe Phe Asn                   275          - #       280          - #       285                      - - gtc tcc atc att ctc ttc ctc aac aag atg ga - #c ctc ctg gtg gag aag          912                                                                       Val Ser Ile Ile Leu Phe Leu Asn Lys Met As - #p Leu Leu Val Glu Lys               290              - #   295              - #   300                          - - gtg aag acc gtg agc atc aag aag cac ttc cc - #g gac ttc agg ggc gac          960                                                                       Val Lys Thr Val Ser Ile Lys Lys His Phe Pr - #o Asp Phe Arg Gly Asp           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - ccg cac cag ctg gag gac gtc cag cgc tac ct - #g gtc cag tgc ttc        gac     1008                                                                    Pro His Gln Leu Glu Asp Val Gln Arg Tyr Le - #u Val Gln Cys Phe Asp                          325  - #               330  - #               335              - - agg aag aga cgg aac cgc agc aag cca ctc tt - #c cac cac ttc acc acc         1056                                                                       Arg Lys Arg Arg Asn Arg Ser Lys Pro Leu Ph - #e His His Phe Thr Thr                       340      - #           345      - #           350                  - - gcc atc gac acc gag aac gtc cgc ttc gtg tt - #c cat gct gtg aaa gac         1104                                                                       Ala Ile Asp Thr Glu Asn Val Arg Phe Val Ph - #e His Ala Val Lys Asp                   355          - #       360          - #       365                      - - acc atc ctg cag gag aac ctg aag gac atc at - #g ctg cag tga                 - #1146                                                                    Thr Ile Leu Gln Glu Asn Leu Lys Asp Ile Me - #t Leu Gln                           370              - #   375              - #   380                          - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR Primer                                            - - <400> SEQUENCE: 2                                                         - - ctcaagggct caagggttct t           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR Primer                                            - - <400> SEQUENCE: 3                                                         - - caggaacatc ccatgcttct c           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR Probe                                             - - <400> SEQUENCE: 4                                                         - - aagcttggca ttccttggca gtattctga         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR Primer                                            - - <400> SEQUENCE: 5                                                         - - gaaggtgaag gtcggagtc             - #                  - #                      - # 19                                                                   - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR Primer                                            - - <400> SEQUENCE: 6                                                         - - gaagatggtg atgggatttc            - #                  - #                      - # 20                                                                   - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR Probe                                             - - <400> SEQUENCE: 7                                                         - - caagcttccc gttctcagcc            - #                  - #                      - # 20                                                                   - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 8                                                         - - ctgagggtcc gcaccacc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 9                                                         - - ggcagcggct gagggtcc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 10                                                        - - agcaggcagc ggctgagg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 11                                                        - - gtcgatgtcg cggctacg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 12                                                        - - ccagcagcgc gtcgatgt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 13                                                        - - gcaggatctt caccaggc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 14                                                        - - ggatgatgcg catctgct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 15                                                        - - cagcagcagg atcttcac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 16                                                        - - gtggacttgc cgctctcg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 17                                                        - - acgtggactt gccgctct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 18                                                        - - gcttgaggaa cgtggact             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 19                                                        - - gcgcatctgc ttgaggaa             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 20                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 20                                                        - - ctcgcggccg tggatgat             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 21                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 21                                                        - - tctggtcgaa ctcgcggc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 22                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 22                                                        - - gcgccttctg gtcgaact             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 23                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 23                                                        - - ctccagcagc gccttctg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 24                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 24                                                        - - tgtcgcggaa ctccagca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 25                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 25                                                        - - tcgaagatgg tgtcgcgg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 26                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 26                                                        - - aggatgttgt cgaagatg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 27                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 27                                                        - - agcccttgag gatgttgt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 28                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 28                                                        - - acaagaaccc ttgagccc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 29                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 29                                                        - - gtgcatcaac aagaaccc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 30                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 30                                                        - - cttatctcgt gcatcaac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 31                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 31                                                        - - actgccaagg aatgccaa             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 32                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 32                                                        - - ttttcagaat actgccaa             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 33                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 33                                                        - - ccatgcttct cattttca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 34                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 34                                                        - - tcaggaacat cccatgct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 35                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 35                                                        - - cgaaggccat caggaaca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 36                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 36                                                        - - ccccgccttg ttctcgaa             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 37                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 37                                                        - - gggacgtaca gctggaag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 38                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 38                                                        - - ctcagggccg ggacgtac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 39                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 39                                                        - - gtgcgctcag ggccggga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 40                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 40                                                        - - cagaatccct ccagagtg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 41                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 41                                                        - - ccctgatgcc agaatccc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 42                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 42                                                        - - gaaagcctcc ctgatgcc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 43                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 43                                                        - - actcgcttct ccggctga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 44                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 44                                                        - - ggaagtactt caccgact             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 45                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 45                                                        - - aagttgtcca ggaagtac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 46                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 46                                                        - - tccggtccaa gttgtcca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 47                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 47                                                        - - ccgatccggt ccaagttg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 48                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 48                                                        - - aattcagctg gccgatcc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 49                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 49                                                        - - actaggaaag taattcag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 50                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 50                                                        - - gatatcttgc ttactagg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 51                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 51                                                        - - cttggtggct ttcctagc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 52                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 52                                                        - - cacaattccc ttggtggc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 53                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 53                                                        - - aagtcatgct ccacaatt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 54                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 54                                                        - - ttaataacga agtcatgc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 55                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 55                                                        - - tcttcttaat aacgaagt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 56                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 56                                                        - - cacatccacc atcttaaa             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 57                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 57                                                        - - ccgctggccg cccacatc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 58                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 58                                                        - - ctggaaccac ttctggcg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 59                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 59                                                        - - ccgtcgaagc actggaac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 60                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 60                                                        - - catgaacagg atggacgt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 61                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 61                                                        - - ctggaggaga ccatgaac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 62                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 62                                                        - - gtcgtactcg ctggagga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 63                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 63                                                        - - ggacctggtc gtactcgc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 64                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 64                                                        - - tccatgagga cctggtcg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 65                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 65                                                        - - ggtgcgcctg tcctccat             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 66                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 66                                                        - - cagccggttg gtgcgcct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 67                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 67                                                        - - ttcatggact ccaccagc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 68                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 68                                                        - - aagatgttca tggactcc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 69                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 69                                                        - - tggtctcgaa gatgttca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 70                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 70                                                        - - ttgttgacga tggtctcg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 71                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 71                                                        - - gttgaagaag agcttgtt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 72                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 72                                                        - - atgatggaga cgttgaag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 73                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 73                                                        - - ggaagagaat gatggaga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 74                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 74                                                        - - gtccatcttg ttgaggaa             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 75                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 75                                                        - - caccaggagg tccatctt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 76                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 76                                                        - - gctcacggtc ttcacctt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 77                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 77                                                        - - agtgcttctt gatgctca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 78                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 78                                                        - - cctgaagtcc gggaagtg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 79                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 79                                                        - - cctgtcgaag cactggac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 80                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 80                                                        - - gttccgtctc ttcctgtc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 81                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 81                                                        - - ggtggaagag tggcttgc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 82                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 82                                                        - - gtggtgaagt ggtggaag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 83                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 83                                                        - - gaagcggacg ttctcggt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 84                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 84                                                        - - catggaacac gaagcgga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 85                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 85                                                        - - ggttctcctg caggatgg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 86                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 86                                                        - - agcatgatgt ccttcagg             - #                  - #                      - #  18                                                                 __________________________________________________________________________

What is claimed is:
 1. An antisense compound 8 to 30 nucleotides inlength targeted to a nucleic acid molecule encoding human G-alpha-12,wherein said antisense compound inhibits the expression of humanG-alpha-12.
 2. The antisense compound of claim 1 which is an antisenseoligonucleotide.
 3. The antisense compound of claim 2 wherein theantisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 4. The antisense compound of claim 3 whereinthe modified internucleoside linkage is a phosphorothioate linkage. 5.The antisense compound of claim 2 wherein the antisense oligonucleotidecomprises at least one modified sugar moiety.
 6. The antisense compoundof claim 5 wherein the modified sugar moiety is a 2'-O-methoxyethylsugar moiety.
 7. The antisense compound of claim 2 wherein the antisenseoligonucleotide comprises at least one modified nucleobase.
 8. Theantisense compound of claim 7 wherein the modified nucleobase is a5-methylcytosine.
 9. The antisense compound of claim 1 wherein theantisense oligonucleotide is a chimeric oligonucleotide.
 10. A method ofinhibiting the expression of human G-alpha-12 in human cells or tissuescomprising contacting said cells or tissues in vitro with the antisensecompound of claim 1 so that expression of human G-alpha-12 is inhibited.